Beta-hairpin peptidomimetics

ABSTRACT

β-Hairpin peptidomimetics of the general formula cyclo(-Tyr 1 -His 2 -Xaa 3 -Cys 4 -Ser 5 -Xaa 6 -DPro 7 -Xaa 8 -Arg 9 -Tyr 10 -Cys 11 -Tyr 12 -Xaa 13 -Xaa 14 -Xaa 15 -Pro 16 -), disulfide bond between Cys 4  and Cys 11 , and pharmaceutically acceptable salts thereof, with Xaa 3 , Xaa 6 , Xaa 8  Xaa 13 , Xaa 14  and Xaa 15  being amino acid residues of certain types which are defined in the description and the claims, have favorable pharmacological properties and can be used for preventing HIV infections in healthy individuals or for slowing and halting viral progression in infected patients; or where cancer is mediated or resulting from CXCR 4  receptor activity; or where immunological diseases are mediated or resulting from CXCR4 receptor activity; or for treating immunosuppression; or during apheresis collections of peripheral blood stem cells and/or as agents to induce mobilization of stem cells to regulate tissue repair. These peptidomimetics can be manufactured by a process which is based on a mixed solid-and solution phase synthetic strategy.

The present invention provides β-hairpin peptidomimetics which arehaving CXCR4 antagonizing activity.

The β-hairpin peptidomimetics of the invention arecyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa_(—-Xaa)¹⁴-Xaa¹⁵-Pro¹⁶-), disulfide bond between Cys⁴ and Cys¹¹, andpharmaceutically acceptable salts thereof, with Xaa³ being Tyr, Tyr(Me)as described herein below or Tyr(CF₃) as described herein below, Xaa⁶being Ala or Acc as described herein below, Xaa⁸ being Orn(iPr) asdescribed herein below, Xaa¹³ being Gln or Glu, Xaa¹⁴ being Lys(iPr) asdescribed herein below, Xaa¹⁵ being ^(D)Pro or ^(D)Lys(iPr) as describedherein below; with the proviso that if Xaa⁶ is Ala, then Xaa¹⁵ is^(D)Lys(iPr) as described herein below.

In addition, the present invention provides an efficient syntheticprocess by which these compounds can, if desired, be made in parallellibrary-format. These β-hairpin peptidomimetics have favorablepharmacological properties and, in addition, show suitable plasmaprotein binding and appropriate clearance rates. Therefore they can beused as active ingredients in low amounts for all kind of drugformulations, in particular extended release drug formulations.

Many medically significant biological processes are mediated by signaltransduction that involves chemokines and their receptors in general andstromal derived factor 1 (SDF-1/CXCL12) and its receptor CXCR4 inparticular.

CXCR4 and its ligand SDF-1 are involved in trafficking of B-cells,hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC).For instance, CXCR4 is expressed on CD34+ cells and has been implicatedin the process of CD34+ cell migration and homing (S. M. Watt, S. P.Forde, Vox sanguinis 2008, 94, 18-32). It has also been shown that theCXCR4 receptor plays an important role in the release of stem andprogenitor cells from the bone marrow to the peripheral blood (L. M.Pelus, S. Fukuda, Leukemia 2008, 22, 466-473). This activity of CXCR4could be very important for efficient apheresis collections ofperipheral blood stem cells. Autologous peripheral blood cells provide arapid and sustained hematopoietic recovery followingauto-transplantation after the administration of high-dose chemotherapyor radiotherapy in patients with haematological malignancies and solidtumors (W. C. Liles et al., Blood 2003, 102, 2728-2730).

Recently, it has been demonstrated that SDF-1 is locally up-regulated inanimal models of injury including focal ischemic stroke, global cerebralischemia, myocardial infarction and hind limb ischemia as well as beinginvolved in recovery after peripheral ischemia or following injury tothe liver, kidney or lung (A. E. Ting, R. W. Mays, M. R. Frey, W. Van'tHof, S. Medicetty, R. Deans, Critical Reviews in Oncology/Hematology2008, 65, 81-93 and literature cited herein; F. Lin, K. Cordes, L. Li,L. Hood, W. G. Couser, S. J. Shankland et al., J. Am. Soc. Nephrol.2003, 14, 1188-1199; C. C. Dos Santos, Intensive Care Med. 2008, 34,619-630). These results suggest that SDF-1 may be a chemoattractant forCXCR4-positive stem cells for tissue and organ repair/regeneration (M.Z. Ratajczak, M. Kucia, R. Reca, M. Majka, A. Janowska-Wieczorek, J.Ratajczak, Leukemia 2004, 18, 29-40). Therefore, modulating theSDF-1/CXCR4 axis by CXCR4 inhibitors should result in a significanttherapeutic benefit by using released stem cells to regulate tissuerepair.

More recently, it has been shown that disrupting the CXCR4/SDF-1 axis byCXCR4 inhibitors plays a crucial role in differential mobilization ofprogenitor cells like HPCs, endothelial (EPCs) and stromal progenitorcells (SPCs) from the bone marrow (S. C. Pitchford, R. C. Furze, C. P.Jones, A. M. Wegner, S. M. Rankin, Cell Stem Cell 2009, 4, 62). Inaddition, bone marrow-derived CXCR4⁺Very Small Embryonic-Like Stem Cells(VSELs) were mobilized in patients with acute myocardial infarctionindicating a hypothetical reparatory mechanism (W. Wojakowski, M.Tendra, M. Kucia, E. Zuba-Surma, E. Paczkowska, J. Ciosek, M. Halasa, M.Król, M. Kazmierski, P. Buszman, A. Ochala, J. Ratajczak, B.Machalinski, M. Z. Ratajczak, J. Am. Coll. Cardiol. 2009, 53, 1). Thesefindings may be exploited to provide efficacious stem cell therapy fortissue regeneration.

Mesenchymal stem cells (MSC) are nonhematopoietic progenitor cellshaving the capability of differentiating into tissues such as bone andcartilage (D. J. Prockop, Science 1997, 276, 71). As a small proportionof MSCs strongly expresses functionally active CXCR4, modulation of theCXCR4/SDF-1 axis may mediate specific migration and homing of thesecells (R. F. Wynn, C. A. Hart, C. Corradi-Perini, L. O'Neill, C. A.Evans, J. E. Wraith, L. J. Fairbaim, I. Bellantuono, Blood 2004, 104,2643).

There is increasing evidence suggesting that chemokines in general andthe SDF-1/CXCR4 interaction in particular play a pivotal role inangiogenesis. Chemokines induce angiogenesis directly by binding theircognate receptors on endothelial cells or indirectly by promotinginflammatory cell infiltrates, which deliver other angiogenic stimuli. Anumber of proinflammatory chemokines including interleukin 8 (IL-8),growth-regulated oncogene, stromal cellderived factor 1 (SDF-1),monocyte chemotactic protein 1 (MCP-1), eotaxin 1, and I-309 have beenshown to act as direct inducers of angiogenesis (X. Chen, J. A. Beutler,T. G. McCloud, A. Loehfelm, L. Yang, H. F. Dong, O. Y. Chertov, R.Salcedo, J. J. Oppenheim, O. M. Howard. Clin. Cancer Res. 2003, 9(8),3115-3123; R. Salcedo, J. J. Oppenheim, Microcirculation 2003, (3-4),359-370).

Recently obtained results show that the CXCR4 receptor is involved inthe chemotactic activity of cancer cells, such as breast cancermetastasis or in metastasis of ovarian cancer (A. Muller, B. Homey, H.Soto, N. Ge, D. Catron, M. E. Buchanan, T. Mc Clanahan, E. Murphey, W.Yuan, S. N. Wagner, J. L. Barrera, A. Mohar, E. Verastegui, A. Zlotnik,Nature 2001, 50, 410; J. M. Hall, K. S. Korach, Molecular Endocrinology2003, 17, 792-803), Non-Hodgin's Lymphoma (F. Bertolini, C. Dell'Agnola,P. Manusco, C. Rabascio, A. Burlini, S. Monestiroli, A. Gobbi, G.Pruneri, G. Martinelli, Cancer Research 2002, 62, 3106-3112), or lungcancer (T. Kijima, G. Maulik, P. C. Ma, E. V. Tibaldi, R. E. Turner, B.Rollins, M. Sattler, B. E. Johnson, R. Salgia, Cancer Research 2002, 62,6304-6311), melanoma, prostate cancer, kidney cancer, neuroblastomia,pancreatic cancer, multiple myeloma, chronic lymphocytic leukemia,hepatocellular carcinoma, colorectal carcinoma, endometrial cancer andgerm cell tumor (H. Tamamura et al., FEBS Letters 2003, 550, 79-83,cited ref.; Z. Wang, Q. Ma, Q. Liu, H. Yu, L. Zhao, S. Shen, J. Yao,British Journal of Cancer 2008, 99, 1695; B. Sung, S. Jhurani, K. S.Ahn, Y. Mastuo, T. Yi, S. Guha, M. Liu, B. Aggarwal, Cancer Res. 2008,68, 8938; H. Liu, Z. Pan, A. Li, S. Fu, Y. Lei, H. Sun, M. Wu, W. Zhou,Cellular and Molecular Immunology, 2008, 5, 373; C. Rubie, O. Kollmar,V. O. Frick, M. Wagner, B. Brittner, S. Graber, M. K. Schilling,Scandinavian Journal of Immunology 2008, 68, 635; S. Gelmini, M.Mangoni, F. Castiglioe, C. Beltrami, A. Pieralli, K. L. Andersson, M.Fambrini, G. I. Taddie, M. Serio, C. Orlando, Clin. Exp. Metastasis2009, 26, 261; D. C. Gilbert, I. Chandler, A. McIntyre, N. C. Goddard,R. Gabe, R. A. Huddart, J. Shipley, J. Pathol. 2009, 217, 94). Blockingthe chemotactic activity with a CXCR4 inhibitor should stop themigration of cancer cells and thus metastasis.

CXCR4 has also been implicated in the growth and proliferation of solidtumors and leukemia/lymphoma. It was shown that activation of the CXCR4receptor was critical for the growth of both malignant neuronal andglial tumors. Moreover, systemic administration of the CXCR4 antagonistAMD3100 inhibits growth of intracranial glioblastoma and medulloblastomaxenografts by increasing apoptosis and decreasing the proliferation oftumor cells O. B. Rubin, A. L Kung, R. S Klein, J. A. Chan, Y. Sun, K.Schmidt, M. W. Kieran, A. D. Luster, R. A. Segal, Proc Natl Acad SciUSA. 2003, 100(23),13513-13518; S. Barbero, R. Bonavia, A. Bajetto, C.Porcile, P. Pirani, J. L. Ravetti, G. L. Zona, R. Spaziante, T. Florio,G. Schettini, Cancer Res. 2003, 63(8), 1969-1974; T. Kijima, G. Maulik,P. C. Ma, E. V. Tibaldi, R. E. Turner, B. Rollins, M. Sattler, B. E.Johnson, R. Salgia. Cancer Res. 2002, 62(21), 6304-6311). CXCR4inhibitors also showed promising in vitro and in vivo efficacies inbreast cancer, small cell lung cancer, pancreatic cancer, gastriccancer, colorectal cancer, malignant melanoma, ovarian cancer,rhabdomyo-sarcoma, prostate cancer as well as chronic lymphocyticleukemia, acute myelogenous leukemia, acute lymphoblastic leukemia,multiple myeloma and Non-Hodgkin's lymphoma (J. A. Burger, A. Peled,Leukemia 2009, 23, 43-52 and literature cited herein).

It is well established that chemokines are involved in a number ofinflammatory pathologies and some of them show a pivotal role in themodulation of osteoclast development. Immunostaining for SDF-1 (CXCL12)on synovial and bone tissue biopsies from both rheumatoid arthritis (RA)and osteoarthritis (OA) samples have revealed strong increases in theexpression levels of chemokines under inflammatory conditions (F.Grassi, S. Cristino, S. Toneguzzi, A. Piacentini, A. Facchini, G.Lisignoli, J. Cell Physiol. 2004; 199(2), 244-251). It seems likely thatthe CXCR4 receptor plays an important role in inflammatory diseases suchas rheumatoid arthritis, asthma, multiple sclerosis, Alzheimer'sdisease, Parkinson's disease, atherosclerosis, or eye diseases such asdiabetic retinopathy and age related macular degeneration (K. R. Shadidiet al., Scandinavian Journal of Immunology 2003, 57, 192-198; J. A.Gonzalo, J. Immunol. 2000, 165, 499-508; S. Hatse et al., FEBS Letters2002, 527, 255-262 and cited references, A. T. Weeraratna, A. Kalehua,I. DeLeon, D. Bertak, G. Maher, M. S. Wade, A. Lustig, K. G. Becker, W.Wood, D. G. Walker, T. G. Beach, D. D. Taub, Exp. Cell Res. 2007, 313,450; M. Shimoji, F. Pagan, E. B. Healton, I. Mocchetti, Neurotox. Res.2009, 16, 318; A. Zernecke, E. Shagdarsuren, C. Weber, Arterioscler.Thromb. Vasc. Biol. 2008, 28, 1897; R. Lima e Silva, J. Shen, S. F.Hackett, S. Kachi, H. Akiyama et al., FASEB 2007, 21, 3219). Themediation of recruitment of immune cells to sites of inflammation shouldbe stopped by a CXCR4 inhibitor.

To date the available therapies for the treatment of HIV infections havebeen leading to a remarkable improvement in symptoms and recovery fromdisease in infected people. Although the highly active anti-retroviraltherapy (HAART) which involves a combination of reversetranscriptase/protease-inhibitor has dramatically improved the clinicaltreatment of individuals with AIDS or HIV infection, there have stillremained several serious problems including multi drug resistance,significant adverse effects and high costs. Particularly desired areanti-HIV agents that block the HIV infection at an early stage of theinfection, such as the viral entry. It has recently been recognized thatfor efficient entry into target cells, human immunodeficiency virusesrequire the chemokine receptors CCR5 and CXCR4 as well as the primaryreceptor CD4 (N. Levy, Engl. J. Med. 1996, 335, 1528-1530). Accordingly,an agent which could block the CXCR4 chemokine receptors should preventinfections in healthy individuals and slow or halt viral progression ininfected patients (J. Cohen, Science 1997, 275, 1261-1264).

Among the different types of CXCR4 inhibitors (M. Schwarz, T. N. C.Wells, A. E. I. Proudfoot, Receptors and Channels 2001, 7, 417-428; Y.Lavrovsky, Y. A. Ivanenkov, K. V. Balakin, D. A. Medvedewa, P. V.Ivachtchenko, Mini Rev. Med. Chem. 2008, 11, 1075-1087), one emergingclass is based on naturally occurring cationic peptide analogues derivedfrom Polyphemusin II which have an antiparallel β-sheet structure, and aβ-hairpin that is maintained by two disulfide bridges (H. Nakashima, M.Masuda, T. Murakami, Y. Koyanagi, A. Matsumoto, N. Fujii, N. Yamamoto,Antimicrobial Agents and Chemoth. 1992, 36, 1249-1255; H. Tamamura, M.Kuroda, M. Masuda, A. Otaka, S. Funakoshi, H. Nakashima, N. Yamamoto, M.Waki, A. Matsumotu, J. M. Lancelin, D. Kohda, S. Tate, F. Inagaki, N.Fujii, Biochim. Biophys. Acta 1993, 209, 1163; WO 95/10534 A1).

Synthesis of structural analogs and structural studies by nuclearmagnetic resonance (NMR) spectroscopy have shown that the cationicpeptides adopt well defined (β-hairpin conformations, due to theconstraining effect of one or two disulfide bridges (H. Tamamura, M.Sugioka, Y. Odagaki, A. Omagari, Y. Kahn, S. Oishi, H. Nakashima, N.Yamamoto, S. C. Peiper, N. Hamanaka, A. Otaka, N. Fujii, Bioorg. Med.Chem. Lett. 2001, 359-362). These results show that the β-hairpinstructure plays an important role in CXCR4 antagonizing activity.

Additional structural studies have indicated that the antagonizingactivity can also be influenced by modulating amphiphilic structure andthe pharmacophore (H. Tamamura, A. Omagari, K. Hiramatsu, K. Gotoh, T.Kanamoto, Y. Xu, E. Kodama, M. Matsuoka, T. Hattori, N. Yamamoto, H.Nakashima, A. Otaka, N. Fujii, Bioorg. Med. Chem. Lett. 2001, 11,1897-1902; H. Tamamura, A. Omagari, K. Hiramatsu, S. Oishi, H.Habashita, T. Kanamoto, K. Gotoh, N. Yamamoto, H. Nakashima, A. Otaka N.Fujii, Bioorg. Med. Chem. 2002, 10, 1417-1426; H. Tamamura, K.Hiramatsu, K. Miyamoto, A. Omagari, S. Oishi, H. Nakashima, N. Yamamoto,Y. Kuroda, T. Nakagawa, A. Otaki, N. Fujii, Bioorg. Med. Chem. Letters2002, 12, 923-928).

The compoundscyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Pro¹⁶-),disulfide bond between Cys⁴ and Cys¹¹, of the invention are cyclicβ-hairpin peptidomimetics exhibiting high CXCR4 antagonizing activity,being useful for efficient apheresis collections of mobilized peripheralblood stern cells and/or using these mobilized cells to regulate tissuerepair, and/or having anti-cancer activity, anti-inflammatory activityand/or anti-HIV activity.

The cyclic β-hairpin conformation is induced by the D-amino acid residue^(D)Pro⁷ and the D-amino acid residue Xaa¹⁵. Further stabilization ofthe hairpin conformation is achieved by the amino acid residues Cys atpositions 4 and 11, which, taken together, form a disulfide bridge.

Surprisingly we have found that the introduction of the basic amino acidresidues Orn(iPr) at position 8, Lys(iPr) at position 14, supported byan optional introduction of ^(D)Lys(iPr) at position 15 ofcyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Pro¹⁶-), disulfide bond between Cys⁴and and Cys¹¹, result in β-hairpin peptidomimetics which have favorablepharmacological properties. These propert ies, combined with suitableplasma protein binding and appropriate clearance rates form apharmacological profile which allows these compounds to be used asactive ingredients in low amounts for all kind of drug formulations, inparticular extended release drug formulations.

The β-hairpin peptidomimetics of the present invention are compounds ofthe general formula

cyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Pro¹⁶-),

disulfide bond between Cys⁴ and Cys¹¹, and pharmaceutically acceptablesalts thereof,

wherein

Xaa³ is Tyr, Tyr(Me), i.e. (2S)-2-amino-(4-methoxyphenyl)-3-propionicacid, or Tyr(CF₃), i.e.(2S)-2-amino-(4-trifluoromethoxyphenyl)-3-propionic acid,

Xaa⁶ is Ala or Acc, the latter being 1-aminocyclopropane-carboxylicacid,

Xaa⁸ is Orn(iPr), i.e. (2S)-N^(ω)-isopropyl-2,5-diaminopentanoic acid,

Xaa¹³ is Gln or Glu,

Xaa¹⁴ is Lys(iPr), i.e. (2S)-N^(ω)-isopropyl-2,6-diaminohexanoic acid,

Xaa¹⁵ is ^(D)Pro, or ^(D)Lys(iPr), i.e.(2R)-N^(ω)-isopropyl-2,6-diaminohexanoic acid, with the proviso that ifXaa⁶ is Ala, then Xaa¹⁵ is ^(D)Lys(iPr).

In a particular embodiment of the present invention the compound iscyclo(-Tyr¹-His²-Tyr³-Cys⁴-Ser⁵-Ala⁶-^(D)Pro⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^(D)Lys(iPr)¹⁵-Pro¹⁶-),disulfide bond between Cys⁴ and Cys¹¹, and pharmaceutically acceptablesalts thereof.

In another particular embodiment of the present invention the compoundiscyclo(-Tyr¹-His²-Tyr³-Cys⁴-Ser⁵-Acc⁶-^(D)Pro⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^(D)Pro₁₅-Pro¹⁶-),disulfide bond between Cys⁴ and Cys¹¹, and pharmaceutically acceptablesalts thereof.

In accordance with the present invention these β-hairpin peptidomimeticscan be prepared by a process which comprises

-   -   (a) coupling an appropriately functionalized solid support with        an appropriately N-protected derivative of Pro which in the        desired end-product is in position 16;    -   (b) removing the N-protecting group from the product thus        obtained;    -   (c) coupling the product thus obtained with an appropriately        N-protected derivative of that amino acid which in the desired        end-product is in position 15, any functional group which may be        present in said N-protected amino acid derivative being likewise        appropriately protected;    -   (d) removing the N-protecting group from the product obtained in        step (c);    -   (e) effecting steps substantially corresponding to steps (c)        and (d) using appropriately N-protected derivatives of amino        acids which in the desired end-product are in positions 14to 1,        any functional group(s) which may be present in said N-protected        amino acid derivatives being likewise appropriately protected;    -   (f) if desired, forming a disulfide bridge between the        side-chains of the Cys residues at position 4 and position 11;        or alternatively, forming the aforesaid linkage subsequent to        step (i), as described herein below;    -   (g) detaching the product thus obtained from the solid support;    -   (h) cyclizing the product cleaved from the solid support;    -   (i) removing any protecting groups present on functional groups        of any members of the chain of amino acid residue; and    -   (j) if desired, attaching one or several isopropyl groups    -   (k) if desired, converting the product thus obtained into a        pharmaceutically acceptable salt or converting a        pharmaceutically acceptable, or unacceptable, salt thus obtained        into the corresponding free compound or into a different,        pharmaceutically acceptable, salt.

The β-hairpin peptidomimetics of this invention can be produced, forexample, by following a procedure comprising the synthesis of the linearpeptide on resin whereas the isopropyl group-bearing amino acidresidue(s) Orn(iPr), Lys(iPr) or ^(D)Lys(iPr) will be incorporated asamino acid building block(s) being commercially available or synthesizedbeforehand; or a procedure comprising the synthesis of a linear peptideon resin and the derivatisation of the amino group-bearing side chainsof amino acid residues protected by acid labile protecting groupssuitable to the Fmoc-based solid phase peptide synthesis strategy bycoupling isopropyl groups in solution at a very late stage of thesynthesis cascade; or following a procedure comprising a suitablecombination of the procedures described before.

The proper choice of the functionalized solid-support (i.e. solidsupport plus linker molecule) and the site of cyclization play key rolesin the synthesis process of the β-hairpin peptidomimetics of theinvention.

The functionalized solid support is conveniently derived frompolystyrene crosslinked with, preferably 1-5%, divinylbenzene;polystyrene coated with polyethyleneglycol spacers (Tentagel®) andpolyacrylamide resins (see also D. Obrecht, J.-M. Villalgordo,“Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries”, Tetrahedron OrganicChemistry Series, Vol. 17, Pergamon, Elsevier Science, 1998).

The solid support is functionalized by means of a linker, i.e. abifunctional spacer molecule which contains on one end an anchoringgroup for attachment to the solid support and on the other end aselectively cleavable functional group used for the subsequent chemicaltransformations and cleavage procedures. For the purposes of the presentinvention two types of linkers are used:

Type 1 linkers are designed to release the amide group under acidicconditions (H. Rink, Tetrahedron Lett. 1987, 28, 3783-3790). Linkers ofthis kind form amides of the carboxyl group of the amino acids; examplesof resins functionalized by such linker structures include4-[(((2,4-dimethoxy-phenyl)Fmoc-aminomethyl)phenoxyacetamido)aminomethyl] PS resin,4-[(((2,4-dimethoxyphenyl)Fmoc-aminomethyl)phenoxy-acetamido)aminomethyl]-4-methyl-benzydrylaminePS resin (Rink amide MBHA PS Resin), and4-[(((2,4-dimethoxyphenyl)Fmoc-aminomethyl)phenoxyacetamido)aminomethyl]benzhydrylaminePS-resin (Rink amide BHA PS resin). Preferably, the support is derivedfrom polystyrene crosslinked with, most preferably 1-5%, divinylbenzeneand functionalized by means of the4-(((2,4-dimethoxy-phenyl)Fmoc-aminomethyl)phenoxyacetamido) linker.

Type 2 linkers are designed to eventually release the carboxyl groupunder acidic conditions. Linkers of this kind form acid-labile esterswith the carboxyl group of the amino acids, usually acid-labile benzyl,benzhydryl and trityl esters; examples of such linker structures include2-methoxy-4-hydroxymethylphenoxy (Sasrin® linker),4-(2,4-dimethoxyphenyl-hydroxymethyl)-phenoxy (Rink linker),4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB linker), trityland 2-chlorotrityl. Preferably, the support is derived from polystyrenecrosslinked with, most preferably 1-5%, divinylbenzene andfunctionalized by means of the 2-chlorotrityl linker.

When carried out as parallel array syntheses the processes of theinvention can be advantageously carried out as described herein belowbut it will be immediately apparent to those skilled in the art howthese procedures will have to be modified in case it is desired tosynthesize one single compound of the invention.

A number of reaction vessels equal to the total number of compounds tobe synthesized by the parallel method are loaded with 25 to 1000 mg,preferably 60 mg, of the appropriate functionalized solid support,preferably 1 to 3% cross-linked polystyrene or Tentagel resin.

The solvent to be used must be capable of swelling the resin andincludes, but is not limited to, dichloromethane (DCM),dimethylformamide (DMF), N-methylpyrrolidone (NMP), dioxane, toluene,tetrahydrofuran (THF), ethanol (EtOH), trifluoroethanol (TFE),isopropylalcohol and the like. Solvent mixtures containing as at leastone component a polar solvent (e. g. 20% TFE/DCM, 35% THF/NMP) arebeneficial for ensuring high reactivity and solvation of the resin-boundpeptide chains (G. B. Fields, C. G. Fields, J. Am. Chem. Soc. 1991, 113,4202-4207).

With the development of various linkers that release the C-terminalcarboxylic acid group under mild acidic conditions, not affectingacid-labile groups protecting functional groups in the side chain(s),considerable progresses have been made in the synthesis of protectedpeptide fragments. The 2-methoxy-4-hydroxybenzylalcohol-derived linker(Sasrin® linker, Mergler et al., Tetrahedron Lett. 1988, 29 4005-4008)is cleavable with diluted trifluoroacetic acid (0.5-1% TFA in DCM) andis stable to Fmoc deprotection conditions during the peptide synthesis,Boc/tBu-based additional protecting groups being compatible with thisprotection scheme. Other linkers which are suitable for the process ofthe invention include the super acid labile4-(2,4-dimethoxyphenyl-hydroxymethyl)-phenoxy linker (Rink linker, H.Rink, Tetrahedron Lett. 1987, 28, 3787-3790), where the removal of thepeptide requires 10% acetic acid in DCM or 0.2% trifluoroacetic acid inDCM; the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid-derived linker(HMPB-linker, Flörsheimer & Riniker, Peptides 1991, 1990 131) which isalso cleaved with 1% TFA/DCM in order to yield a peptide fragmentcontaining all acid labile side-chain protective groups; and, inaddition, the 2-chlorotritylchloride linker (Barlos et al., TetrahedronLett. 1989, 30, 3943-3946), which allows the peptide detachment using amixture of glacial acetic acid/trifluoroethanol/DCM (1:2:7) for 30 min.

Suitable protecting groups for amino acids and, respectively, for theirresidues are, for example,

-   -   for the amino group (as is present e.g. also in the side-chain        of lysine or ornithine)    -   Cbz benzyloxycarbonyl    -   Boc tert-butyloxycarbonyl    -   Fmoc 9-fluorenylmethoxycarbonyl    -   Alloc allyloxycarbonyl    -   Teoc tri methylsilylethoxycarbonyl    -   Tcc trichloroethoxycarbonyl    -   Nps o-nitrophenylsulfonyl;    -   Trt triphenymethyl or trityl    -   ivDde (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl    -   for the carboxyl group (as is present e. g. also in the        side-chain of glutamic acid) by conversion into esters with the        alcohol components    -   tBu tert-butyl    -   Bn benzyl    -   Me methyl    -   Ph phenyl    -   Pac phenacyl        -   allyl    -   Tse trimethylsilylethyl    -   Tce trichloroethyl;    -   ivDde (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl    -   for the guanidino group (as is present e. g. in the side-chain        of arginine)    -   Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl    -   Ts tosyl (i. e. p-toluenesulfonyl)    -   Cbz benzyloxycarbonyl    -   Pbf pentamethyldihydrobenzofuran-5-sulfonyl    -   for the hydroxy group (as is present e. g. in the side-chain of        serine)    -   tBu tert-butyl    -   Bn benzyl    -   Trt trityl    -   Alloc allyloxycarbonyl    -   and for the mercapto group (as is present e. g. in the        side-chain of cysteine)    -   Acm acetamidomethyl    -   tBu tert-butyl    -   Bn benzyl    -   Trt trityl    -   Mtr 4-methoxytrityl.

The 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid derivativesare preferably used as the building blocks for the construction of theβ-hairpin loop mimetics of the invention. For the deprotection, i. e.cleaving off of the Fmoc group, 20% piperidine in DMF or 2% DBU/2%piperidine in DMF can be used.

The linkage of isopropyl groups to amino group-bearing side chains of9-fluorenylmethoxycarbonyl (Fmoc) -protected amino acid derivatives toform isopropylated amino group-bearing side chains of (Fmoc) -protectedamino acid derivatives is known in the art. The procedure forintroducing an isopropyl group can be accomplished e.g. by reductivealkylation e.g. treatment of the amino group of the amino group-bearingside chain of an amino acid building block like e.g. Orn with acetone inthe presence of a suitable reducing agent like e.g. sodiumtriacetoxyborohydride. Protecting groups like e.g Boc suitable forispropylated amino group-bearing side chains of (Fmoc) -protected aminoacid derivatives can be introduced by subsequent reaction withdi-tert-butyl dicarbonate in the presence of a base such as sodiumbicarbonate.

The quantity of the reactant, i.e. of the amino acid derivative, isusually 1 to 20 equivalents based on the milliequivalents per gram(meq/g) loading of the functionalized solid support (typically 0.1 to2.85 meq/g for polystyrene resins) originally weighed into the reactiontube. Additional equivalents of reactants can be used, if required, todrive the reaction to completion in a reasonable time. The preferredworkstations (without, however, being limited thereto) are Labsource'sCombi-chem station, Protein Technologies' Symphony and MultiSynTech's-Syro synthesizer, the latter additionally equipped with atransfer unit and a reservoir box during the process of detachment ofthe fully protected linear peptide from the solid support. Allsynthesizers are able to provide a controlled environment, for example,reactions can be accomplished at temperatures different from roomtemperature as well as under inert gas atmosphere, if desired.

Amide bond formation requires the activation of the a-carboxyl group forthe acylation step. When this activation is being carried out by meansof the commonly used carbodiimides such as dicyclohexylcarbodiimide(DCC, Sheehan & Hess, J. Am. Chem. Soc. 1955, 77, 1067-1068) ordiisopropylcarbodiimide (DIC, Sarantakis et al Biochem. Biophys. Res.Commun. 1976, 73, 336-342), the resulting dicyclohexylurea and,respectively, diisopropylurea is insoluble and, respectively, soluble inthe solvents generally used. In a variation of the carbodiimide method1-hydroxybenzotriazole (HOBt, Konig & Geiger, Chem. Ber. 1970, 103,788-798) is included as an additive to the coupling mixture. HOBtprevents dehydration, suppresses racemization of the activated aminoacids and acts as a catalyst to improve the sluggish coupling reactions.Certain phosphonium reagents have been used as direct coupling reagents,such as benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP, Castro et al., Tetrahedron Lett. 1975, 14,1219-1222; Synthesis 1976, 751-752), orbenzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexaflurophoshate(Py-BOP, Coste et al., Tetrahedron Lett. 1990, 31, 205-208), or2-(1H-benzotriazoll-yl-) 1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), or hexafluorophosphate (HBTU, Knorr et al., Tetrahedron Lett.1989, 30, 1927-1930); these phosphonium reagents are also suitable forin situ formation of HOBt esters with the protected amino acidderivatives. More recently diphenoxyphosphoryl azide (DPPA) orO-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TATU) orO-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU)/7-aza-1-hydroxy benzotriazole (HOAt, Carpinoet al., Tetrahedron Lett. 1994, 35, 2279-2281) or-(6-Chloro-1H-benzotriazol-1-yl-)-N,N,N′,N′-1,1,3,3-tetramethyl-Uroniumtetrafluoroborate (TCTU), or hexafluorophosphate (HCTU, Marder, Shivoand Albericio: HCTU and TCTU: New Coupling Reagents: Development andIndustrial Applications, Poster Presentation, Gordon Conference February2002) have also been used as coupling reagents as well as1,1,3,3-bis(tetramethylene)chlorouronium hexafluoro-phosphate (PyCIU)especially for coupling N-methylated amino acids (J. Coste, E. Frerot,P. Jouin, B. Castro, Tetrahedron Lett. 1991, 32, 1967) orpentafluorophenyl diphenyl-phosphinate (S. Chen, J. Xu, TetrahedronLett. 1991, 32, 6711).

Due to the fact that near-quantitative coupling reactions are essential,it is desirable to have experimental evidence for completion of thereactions. The ninhydrin test (Kaiser et al., Anal. Biochemistry 1970,34, 595), where a positive colorimetric response to an aliquot ofresin-bound peptide indicates qualitatively the presence of the primaryamine, can easily and quickly be performed after each coupling step.Fmoc chemistry allows the spectrophotometric detection of the Fmocchromophore when it is released with the base (Meienhofer et al., Int.J. Peptide Protein Res. 1979, 13, 35-42).

The resin-bound intermediate within each reaction vessel is washed freeof excess of retained reagents, of solvents, and of by-products byrepetitive exposure to pure solvent(s) by one of the two followingmethods:

1) The reaction vessels are filled with solvent (preferably 5 mL),agitated for 5 to 300 minutes, preferably 15 minutes, and drained toexpel the solvent;

2) The reaction vessels are filled with solvent (preferably 5 mL) anddrained into a receiving vessel such as a test tube or vial.

Both of the above washing procedures are repeated up to about 50 times(preferably about 10 times), monitoring the efficiency of reagent,solvent, and by-product removal by methods such as TLC, GC, orinspection of the washings.

The above described procedure of reacting the resin-bound compound withreagents within the reaction tubes followed by removal of excessreagents, by-products, and solvents is repeated with each successivetransformation until the final resin-bound fully protected linearpeptide has been obtained.

Before this fully protected linear peptide is detached from the solidsupport, a disulfide bridge between Cys⁴ and Cys¹¹ can be formed.

For the formation of a disulfide bridge preferably a solution of 10equivalents of iodine solution is applied in DMF or in a mixture ofCH₂Cl₂/MeOH for 1.5 h which is repeated for another 3 h with a freshiodine solution after filtering of the iodine solution, or in a mixtureof DMSO and acetic acid solution, buffered with 5% NaHCO₃ to pH 5-6 for4 h, or in water after adjusting to pH 8 with ammonium hydroxidesolution by stirring for 24 h, or in a solution of NMP andtri-n-butylphosphine (preferably 50 eq.).

Alternatively, the formation of the disulfide bridge between Cys⁴ andCys¹¹ can be carried out subsequent to the work-up method 2), asdescribed herein below, by stirring the crude fully deprotected andcyclized peptide for 24 h in water containing DMSO up to 15% by volume,buffered with 5% NaHCO₃ to pH 5-6, or buffered with ammonium acetate topH 7-8, or adjusted with ammonium hydroxide to pH 8. Followingevaporation to drynesscyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Pro¹⁶-),disulfide bond between Cys⁴ and Cys¹¹, is obtained as end-product.

Detachment of the fully protected linear peptide from the solid supportis achieved by exposing the loaded resin with a solution of the reagentused for cleavage (preferably 3 to 5 mL). Temperature control,agitation, and reaction monitoring are implemented as described above.Via a transfer-unit the reaction vessels are connected with a reservoirbox containing reservoir tubes to efficiently collect the cleavedproduct solutions. The resins remaining in the reaction vessels are thenwashed 2 to 5 times as above with 3 to 5 mL of an appropriate solvent toextract (wash out) as much of the detached products as possible. Theproduct solutions thus obtained are combined, taking care to avoidcross-mixing. The individual solutions/extracts are then manipulated asneeded to isolate the final compounds. Typical manipulations include,but are not limited to, evaporation, concentration, liquid/liquidextraction, acidification, basification, neutralization or additionalreactions in solution.

The solutions containing fully protected linear peptide derivativeswhich have been cleaved off from the solid support and neutralized witha base, are evaporated. Cyclization is then effected in solution usingsolvents such as DCM, DMF, dioxane, THF and the like. Various couplingreagents which were mentioned earlier can be used for the cyclization.The duration of the cyclization is about 6-48 h, preferably about 16 h.The progress of the reaction is followed, e. g. by RP-HPLC (ReversePhase High Performance Liquid Chromatography). Then the solvent isremoved by evaporation, the fully protected cyclic peptide derivative isdissolved in a solvent which is not miscible with water, such as DCM,and the solution is extracted with water or a mixture of water-misciblesolvents, in order to remove any excess of the coupling reagent.

Finally, the fully protected peptide derivative is treated with 95% TFA,2.5% H₂O, 2.5% TIS or another combination of scavengers for effectingthe cleavage of protecting groups. The cleavage reaction time iscommonly 30 minutes to 12 h, preferably about 2.5 h.

Alternatively, the detachment and complete deprotection of the fullyprotected peptide from the solid support can be achieved manually inglass vessels.

After full deprotection, for example, the following methods can be usedfor further work-up:

1) The volatiles are evaporated to dryness and the crude peptide isdissolved in 20% AcOH in water and extracted with isopropyl ether orother solvents which are suitable therefor. The aqueous layer iscollected and evaporated to dryness, and the fully deprotected cyclicpeptide,cyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Pro¹⁶-),disulfide bond between Cys⁴ and Cys¹¹, is obtained as final product;

2) The deprotection mixture is concentrated under vacuum. Followingprecipitation of the fully deprotected peptide in diethylether atpreferably 0° C. the solid is washed up to about 10 times, preferably 3times, dried, and the fully deprotected cyclic peptide,cyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Pro¹⁶-)disulfidebond between Cys⁴ and Cys¹¹, is obtained as final product, if adisulfide bond between Cys⁴ and Cys¹¹ has been formed on solid supportas described herein above.

If the above mentioned orthogonal protecting group strategy forintroducing one or more isopropyl groups in solution has been followed,then all amino groups of amino acid residues formerly protected by acidlabile protecting groups have been liberated at this stage of thesynthesis cascade. Thus, it is possible, if desired, to couple anisopropyl group. This coupling can be accomplished by applying e.g. areductive alkylation using acetone in the presence of a suitablereducing agent like e.g. sodium cyano borhydride. Thus, for example, thepeptide is dissolved in MeOH (4.4 mM) containing acetic acid (0.2 M).After adding an excess of acetone (780 eq) the reaction mixture iscompleted with a solution of sodium cyano borhydride in MeOH (0.6 M; 1.3eq per isopropyl group desired to be introduced) and vigorously shakenat room temperature. Following completion of the conversion monitored byLC-MS, water is added and the solvents are evaporated.

As mentioned earlier, it is thereafter possible, if desired, to convertthe fully deprotected cyclic product thus obtained into apharmaceutically acceptable salt or to convert a pharmaceuticallyacceptable, or unacceptable, salt thus obtained into the correspondingfree compound, or into a different, pharmaceutically acceptable, salt.Any of these operations can be carried out by methods well known in theart.

The β-hairpin peptidomimetics of the invention can be used in a widerange of applications in order to prevent HIV infections in healthyindividuals and slow or halt viral progression in infected patients, orwhere cancer is mediated or resulting from the CXCR4 receptor activity,or where immunological diseases are mediated or resulting from CXCR4receptor activity; or these β-hairpin peptidomimetics can be used totreat immunosuppression, or they can be used during apheresiscollections of peripheral blood stem cells and/or as agents to inducemobilization of stem cells to regulate tissue repair.

The β-hairpin peptidomimetics of the invention may be administered perse or may be applied as an appropriate formulation together withcarriers, diluents or excipients well known in the art.

When used to treat or prevent HIV infections or cancer such as breastcancer, brain cancer, prostate cancer, heptatocellular carcinoma,colorectal cancer, lung cancer, kidney cancer, neuroblastoma, ovariancancer, endometrial cancer, germ cell tumor, eye cancer, multiplemyeloma, pancreatic cancer, gastric cancer, rhabdomyo-sarcoma, melanoma,chronic lyphomphocytic leukemia, acute myelogenous leukemia, acutelymphoblastic leukemia, multiple myeloma and Non-Hodgkin's lymphoma;metastasis, angiogenesis, and haematopoetic tissues; or inflammatorydisorders such as asthma, allergic rhinitis, hypersensitivity lungdiseases, hypersensitivity pneumonitis, eosinophilic pneumonias,delayed-type hypersensitivity, interstitial lung disease (ILD),idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis,systemic lupus erythematosus, ankylosing sponylitis, systemic sclerosis,Sjogren's syndrome, systemic anaphylaxis or hypersensitivity responses,drug allergies, rheumatoid arthritis, psoriatic arthritis, multiplesclerosis, Alzheimer's disease, Parkinson's disease, atherosclerosis,myasthenia gravis, juvenile onset diabetes, glomerulonephritis,autoimmune throiditis, graft rejection, including allograft rejection orgraft-versus-host disease, inflammatory bowel diseases and inflammatorydermatoses; or to treat eye diseases like glaucoma, diabethicretinopathy and age related macular degeneration; or to treat focalischemic stroke, global cerebral ischemia, myocardial infarction, hindlimb ischemia or peripheral ischemia; or to treat injury of the liver,kidney or lung; or to treat immunosuppression, includingimmunosuppression induced by chemotherapy, radiation therapy orgraft/transplantation rejection, the β-hairpin peptidomimetics of theinvention can be administered singly, as mixtures of several β-hairpinpeptidomimetics, in combination with other anti-HIV agents, orantimicrobial agents or anti-cancer agents or anti-inflammatory agents,or in combination with other pharmaceutically active agents. Theβ-hairpin peptidomimetics of the invention can be administered per se oras pharmaceutical compositions.

Pharmaceutical compositions comprising β-hairpin peptidomimetics of theinvention may be manufactured by means of conventional mixing,dissolving, granulating, coated tablet-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients orauxilliaries which facilitate processing of the active β-hairpinpeptidomimetics into preparations which can be used pharmaceutically.Proper formulation depends upon the method of administration chosen.

For topical administration the β-hairpin peptidomimetics of theinvention may be formulated as solutions, gels, ointments, creams,suspensions, powders etc. as are well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injections, the β-hairpin peptidomimetics of the invention may beformulated in adequate solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline buffer. The solutions may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the β-hairpin peptidomimetics of the invention may be inpowder form for combination with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation as known in the art.

For oral administration, the compounds can be readily formulated bycombining the active β-hairpin peptidomimetics of the invention withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the β-hairpin peptidomimetics of the invention to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions, powders etc., for oral ingestion by a patient tobe treated. For oral formulations such as, for example, powders,capsules and tablets, suitable excipients include fillers such assugars, such as lactose, sucrose, mannitol and sorbitol; cellulosepreparations such as maize starch, wheat starch, rice starch, potatostarch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone(PVP); granulating agents; and binding agents. If desired,desintegrating agents may be added, such as cross-linkedpolyvinylpyrrolidones, agar, or alginic acid or a salt thereof, such assodium alginate. If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. In addition, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the composition may take the form of tablets,lozenges, etc. formulated as usual.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories together with appropriate suppository bases suchas cocoa butter or other glycerides.

In addition to the formulations described above, the β-hairpinpeptidomimetics of the invention may also be formulated as depotpreparations. Such long acting formulations may be administered byimplantation (e.g. subcutaneously or intramuscularly) or byintramuscular injection. For the manufacture of such depot preparationsthe β-hairpin peptidomimetics of the invention may be formulated withsuitable polymeric or hydrophobic materials (e.g. as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly soluble salts.

In addition, other pharmaceutical delivery systems may be employed suchas liposomes and emulsions well known in the art. Certain organicsolvents such as dimethylsulfoxide may also be employed. Additionally,the β-hairpin peptidomimetics of the invention may be delivered using asustained-release system, such as semipermeable matrices of solidpolymers containing the therapeutic agent (e.g. for coated stents).Various sustained-release materials have been established and are wellknown by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the therapeutic agent, additional strategies forprotein stabilization may be employed.

As the β-hairpin peptidomimetics of the invention contain chargedresidues, they may be included in any of the above describedformulations as such or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts tend to be more soluble in aqueous andother protic solvents than are the corresponding free forms.Particluarly suitable pharmaceutically acceptable salts include saltswith carboxylic, phosphonic, sulfonic and sulfamic acids, e.g. aceticacid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid,glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid,pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid,citric acid, amino acids, such as glutamic acid or aspartic acid, maleicacid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid,adamantanecarboxylic acid, benzoic acid, salicylic acid,4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid,cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonicacid, ethane-1,2-disulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid, 1,5-napthalenedisulfonic acid, 2-, 3- or4-methyl-benzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid,dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- orN-propyl-sulfamic acid, and other organic protonic acids, such asascorbic acid. Suitable inorganic acids are for example hydrohalicacids, such as hydrochloric acid, sulfuric acid and phosphoric acid.

The β-hairpin peptidomimetics of the invention, or compositions thereof,will generally be used in an amount effective to achieve the intendedpurpose. It is to be understood that the amount used will depend on aparticular application.

For topical administration to treat or prevent HIV infections atherapeutically effective dose can be determined using, for example, thein vitro assays provided in the examples. The treatment may be appliedwhile the HIV infection is visible, or even when it is not visible. Anordinary skilled expert will be able to determine therapeuticallyeffective amounts to treat topical HIV infections without undueexperimentation.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating β-hairpinpeptidomimetic concentration range that includes the IC₅₀ as determinedin the cell culture. Such information can be used to more accuratelydetermine useful doses in humans.

Initial dosages can also be determined from in vivo data, e.g. animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amounts for applications as anti-HIV agents may be adjustedindividually to provide plasma levels of the β-hairpin peptidomimeticsof the invention which are sufficient to maintain the therapeuticeffect. Therapeutically effective serum levels may be achieved byadministering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the β-hairpin peptidomimetics of the inventionmay not be related to plasma concentration. One having the ordinaryskill in the art will be able to optimize therapeutically effectivelocal dosages without undue experimentation.

The amount of β-hairpin peptidomimetics administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgement of the prescribing physician.

The anti-HIV therapy may be repeated intermittently while infections aredetectable or even when they are not detectable. The therapy may beprovided alone or in combination with other drugs, such as for exampleother anti-HIV agents or anti-cancer agents, or other antimicrobialagents.

Normally, a therapeutically effective dose of the β-hairpinpeptidomimetics described herein will provide therapeutic benefitwithout causing substantial toxicity. Toxicity of the β-hairpinpeptidomimetics of the invention can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in humans. Thedosage of the β-hairpin peptidomimetics of the invention lies preferablywithin a range of circulating concentrations that include the effectivedose with little or no toxicity. The dosage may vary within the rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dose can bechosen by the individual physician in view of the patient's condition(see, e.g. Fingl et al. 1975, In: The Pharmacological Basis ofTherapeutics, Ch. 1, p. 1).

The present invention may also include compounds, which are identical tothe compounds of the general formulacyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa^(l-5)-Pro¹⁶-),disulfide bond between Cys⁴ and Cys¹¹, except that one or more atoms arereplaced by an atom having an atomic mass number or mass different fromthe atomic mass number or mass usually found in nature, e.g. compoundsenriched in ²H (D), ³H, ¹¹C, ¹⁴C, ¹²⁹I etc. These isotopic analogs andtheir pharmaceutical salts and formulations are considered useful agentsin the therapy and/or diagnostic, for example, but not limited to, wherea fine-tuning of in vivo half-life time could lead to an optimizeddosage regimen.

The following Examples illustrate the present invention but are not tobe construed as limiting its scope in any way.

EXAMPLES

1. Peptide Synthesis

Coupling of the First Protected Amino Acid Residue to the Resin

1 g (1.4 mMol) 2-chlorotritylchloride resin (1.4 mMol/g; 100-200 mesh,copoly(styrene-1% DVB) polymer matrix; Barlos et al. Tetrahedron Lett.1989, 30, 3943-3946) was filled into a dried flask. The resin wassuspended in CH₂Cl₂ (5 mL) and allowed to swell at room temperatureunder constant shaking for 30 min. A solution of 0.98 mMol (0.7 eq) ofthe first suitably protected amino acid residue (see below) in CH₂Cl₂ (5mL) mixed with 960 μl (4 eq) of diisopropylethylamine (DIEA) was added.After shaking the reaction mixture for 4 h at 25° C., the resin wasfiltered off and washed successively with CH₂Cl₂ (1×), DMF (1×) andCH₂Cl₂ (1×). A solution of CH₂Cl₂/MeOH/DIEA (17/2/1, 10 mL) was added tothe resin and the suspension was shaken for 30 min. After filtration theresin was washed in the following order with CH₂Cl₂ (1×), DMF (1×),CH₂Cl₂ (1×), MeOH (1×), CH₂Cl₂ (1×), MeOH (1×), CH₂Cl₂ (2×), Et₂O (2×)and dried under vacuum for 6 hours.

Loading was typically 0.6-0.7 mMol/g.

The following preloaded resins was prepared:

Fmoc-Pro-O-2-chlorotrityl resin.

The synthesis was carried out employing a Syro-peptide synthesizer(MultiSynTech) using 24-96 reaction vessels. In each vessel 0.04 mMol ofthe above resin was placed and the resin was swollen in CH₂Cl₂ and DMFfor 15 min, respectively. The following reaction cycles were programmedand carried out:

Step Reagent Time 1 DMF, wash 2 × 1 min 2 20% piperidine/DMF 1 × 5 min,1 × 15 min 3 DMF, wash 5 × 1 min 4 5 eq Fmoc amino acid/DMF + 5 1 × 60min  eq Py-BOP/DMF, 10 eq DIEA/DMF 5 DMF, wash 3 × 1 min

Step 4 was repeated once.

Unless indicated otherwise, the final coupling of an amino acid wasfollowed by Fmoc deprotection by applying steps 1-3 of the abovedescribed reaction cycle.

Amino Acid Building Block Syntheses

Synthesis of Fmoc-Orn(iPr,Boc)-OH

The synthesis of (2S)-N^(α)-fluorenylmethoxylcarbonyl-N^(ω),N^(ω)-tert-butyloxycarbonyl-isopropyl-2,5-diaminopentanoicacid was accomplished by suspending 15.2 g Fmoc-Orn-OH*HCl in 150 mL THF(0.26 M) followed by adding 375 mL acetone (132 eq) and 20.6 g sodiumtriacetoxyborohydride (2.5 eq). The reaction mixture was stirred for 2 hand subsequent to completion of the reaction (monitored by LC-MS) 120 mLof sat. Na₂CO₃-solution and 10.2 g Boc₂O (1.2 eq) were added. Afterstirring overnight sat. Na₂CO₃-solution and Boc₂O were added again twicein portions according to the remaining starting material. Followingcompletion of the Boc-introduction hexane was added twice, separated,and the aqueous layer was acidified with 5 N HCl_(aq) (pH=1) andextracted thrice with ethyl acetate thereafter. Finally, the combinedorganic layers were dried with Na₂SO₄ and evaporated to obtain theproduct as white foam.

The amino acid building blocks Fmoc-^(D)Lys(iPr,Boc)-OH andFmoc-Lys(iPr,Boc)-OH could be synthesized accordingly; the latter isalso commercially available.

Cyclization and Work Up of Backbone Cyclized Peptides

Cleavage of the Fully Protected Peptide Fragment

After completion of the synthesis, the resin (0.04 mMol) was suspendedin 1 mL (0.13 mMol, 3.4 eq) of 1% TFA in CH₂Cl₂ (v/v) for 3 minutes,filtered, and the filtrate was neutralized with 1 mL (0.58 mMol, 14.6eq) of 10% DIEA in CH₂Cl₂ (v/v). This procedure was repeated three timesto ensure completion of the cleavage. The filtrate was evaporated todryness and a sample of the product was fully deprotected by using acleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% waterand 2.5% triisopropylsilane (TIS) to be analyzed by reverse phase-HPLC(C₁₈ column) and ESI-MS to monitor the efficiency of the linear peptidesynthesis.

Cyclization of the Fully Protected Linear Peptide

The fully protected linear peptide (0.04 mMol) was dissolved in DMF (4μMol/mL). Then 30.4 mg (0.08 mMol, 2 eq) of HATU, 10.9 mg (0.08 mMol, 2eq) of HOAt and 28 μl (0.16 mMol, 4 eq) DIEA were added, and the mixturewas vortexed at 25° C. for 16 hours and subsequently concentrated underhigh vacuum. The residue was partitioned between CH₂Cl₂ and H₂O/CH₃CN(90/10: v/v). The CH₂Cl₂ phase was evaporated to yield the fullyprotected cyclic peptide.

Full Deprotection of the Cyclic Peptide

The fully protected cyclic peptide obtained was dissolved in 3 mL of thecleavage mixture containing 82.5% trifluoroacetic acid (TFA), 5% water,5% thioanisole, 5% phenol and 2.5% ethanedithiole (EDT). The mixture wasallowed to stand at 25° C. for 2.5 hours and thereafter concentratedunder vacuum. After precipitation of the cyclic fully deprotectedpeptide in diethylether (Et₂O) at 0° C. the solid was washed twice withEt₂O and dried.

Formation of Disulfide β-Astrand Linkage and Purification

After full deprotection, the crude peptide was dissolved in 0.1 Mammonium acetate buffer (1 mg/1 mL, pH=7-8). DMSO (up to 5% by volume)was added and the solution was shaken overnight. Following evaporationthe residue was purified by preparative reverse phase HPLC.

After lyophilisation the products were obtained as white powders andanalysed by the following analytical method: Analytical HPLC retentiontimes (RT, in minutes) were determined using a Ascentis Express C18column, 50×3.0 mm, (cod. 53811-U-Supelco) with the following solvents A(H₂O+0.1% TFA) and B (CH₃CN+0.1% TFA) and the gradient: 0-0.05 min: 97%A, 3% B; 3.4 min: 33% A 67% B; 3.41-3.65 min: 3% A, 97% B; 3.66-3.7 min:97% A, 3% B. Flow rate=1.3 mL/min; UV_Vis=220 nm.

Example 1

Starting resin was Fmoc-Pro-O-2-chlorotrityl resin, which was preparedas described above. To that resin ^(D)Lys(iPr), finally at position 15,was grafted. The linear peptide was synthesized on solid supportaccording to the procedure described above in the following sequence:

Resin-Pro¹⁶-^(D)(iPr)¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Orn(iPr)⁸-^(D)Pro⁷-Ala⁶-Ser⁵-Cys⁴-Tyr³-His²-Tyr¹.Following a final Fmoc deprotection as described above, the peptide wascleaved from the resin, cyclized, deprotected and, after formation ofthe disulfide β-strand linkage as described above, purified as indicatedabove.

The HPLC-retention time (minutes) was determined using the analyticalmethod as described above (UV-purity [after preparative HPLC]: 95%; RT:1.54; [M+3H]/3=709.9).

Example 2

Starting resin was Fmoc-Pro-O-2-chlorotrityl resin, which was preparedas described above. To that resin ^(D)Pro, finally at position 15, wasgrafted. The linear peptide was synthesized on solid support accordingto the procedure described above in the following sequence:

Resin-Pro¹⁶-^(D)Pro¹⁵-Lys(iPr)¹⁴-Gln¹³-Tyr¹²-Cys¹¹-Tyr¹⁰-Arg⁹-Orn(iPr)⁸-^(D)Pro⁷-Acc⁶-Ser⁵-Cys⁴-Tyr³-His²⁻Tyr¹.Following a final Fmoc deprotection as described above, the peptide wascleaved from the resin, cyclized, deprotected and, after formation ofthe disulfide β-strand linkage as described above, purified as indicatedabove.

The HPLC-retention time (minutes) was determined using the analyticalmethod as described above (UV-purity [after preparative HPLC]: 95%; RT:1.58; [M+3H]/3=689.3).

2. Biological Methods

2.1. Preparation of the Peptides

Lyophilized peptides were weighed on a Microbalance (Mettler MT5) anddissolved in DMSO to a final concentration of 10 mM. Stock solutionswere kept at +4° C., light protected. The biological assays were carriedout under assay conditions having less than 1% DMSO unlike indicatedotherwise.

2.2. Cell Culture

Namalwa cells (CXCR4 natively expressing non-adherent cells, ATCCCRL-1432) were cultured in RPMI1640 plus 10% FBS, and pen/strept. HELAcells were maintained in RPMI1640 plus 10% FBS, pen/strept and 2 mML-glutamine. Cos-7 cells were grown in DMEM medium with 4500 mg/mLglucose supplemented with 10% FCS, pen/strept and 2 mM L-glutamine. Allcell lines were grown at 37° C. at 5% CO₂. Cell media, mediasupplements, PBS-buffer, HEPES, antibiotic/antimycotic, pen/strept, nonessential amino acid, L-glutamine, β-mercaptoethanol and sera werepurchased from Gibco (Pailsey, UK). All fine chemicals were supplied byMerck (Darmstadt, Germany).

2.3. Chemotactic Assay (Cell Migration Assay)

The chemotactic response of Namalwa cells (ATCC CRL-1432) to a gradientof stromal cell-derived factor 1α (SDF-1) was measured using a modifiedBoyden chamber chemotaxis system (ChemoTx; Neuroprobe). In this system,the upper chamber of each well is separated from the lower chambercontaining the chemoattractant SDF-1 by a polycarbonate membrane (5 μmpore size). A circular area of that membrane in the region that coverseach lower well is enclosed by a hydrophobic mask to retain the cellsuspension within this area. The system was prepared by loading thebottom wells with aliquots of 30 μL of chemotaxis medium (RPMI 1640without Phenol red+0.5% BSA) comprising either appropriate serialdilutions of peptides or no peptide at all in combination with SDF-1(0.9 nM) or without the chemoattractant. The membrane was placed overthe bottom wells, and aliquots of 50 μL of a suspension of Namalwa cells(3.6×10⁶ cells/mL) in chemotaxis medium, premixed with chemotaxis mediumcomprising either appropriate serial dilutions of peptides or no peptideat all, was delivered onto each of the hydrophobically limited regionsof the upper surface of the membrane. The cells were allowed to migrateinto the bottom chamber for 5 h at 37° C. in 5% CO₂. After this period,the membrane was removed and its topside was carefully wiped and washedwith PBS to eliminate non-migrated cells. Migrated cells weretransferred using a “funnel” adaptor to a receiving 96-well plate andthe cell number was determined by using the CyQuant™ NF cellproliferation assay (Invitrogen) based on the measurement of cellularDNA content via fluorescent dye binding. Following the manufacturer'sdirections, 50 μL of CyQuant™ dye reagent/HBSS buffer (1/500 [v/v]) wereadded to each well of the above mentioned receiving 96-well plate. Afterincubation for 0.5 h at room temperature the plate was sealed and thefluorescence intensity of each sample was measured by using a Wallac1420 VICTOR²™ plate reader (PerkinElmer) with excitation at 485 nm andemission detection at 535 nm. Finally, the data were normalized by usingthe controls and IC₅₀-values values were determined using GraphPadPrism™ (GraphPad) by fitting a logarithmic curve to the averageddatapoints.

2.4. Cytotoxicity Assay

The cytotoxicity of the peptides to HELA cells (Acc57) and COS-7 cells(CRL-1651) was determined using the MTT reduction assay (T. Mossman, J.Immunol. Meth. 1983, 65, 55-63; M. V. Berridge, A. S. Tan, Arch.Biochem. Biophys. 1993, 303, 474-482). Briefly, the method was asfollows: 4000 HELA cells/well and 3400 COS-7 cells/well were seeded andgrown in 96-well microtiter plates for 24 h at 37° C. at 5% CO₂.Thereafter, time zero (Tz) was determined by MTT reduction (see below).The supernatant of the remaining wells was discarded, and fresh mediumand compounds in serial dilutions (12.5, 25 and 50 μM, triplicates; 0μM, blank) were pipetted into the wells. After incubation of the cellsfor 48 h at 37° C. at 5% CO₂ the supernatant was discarded again and 100μL MTT reagent (0.5 mg/mL in RPMI1640 and DMEM, respectively)/well wasadded. Following incubation at 37° C. for 2-4 h the media were aspiratedand the cells were spiked (100 μL isopropanol/well). The absorbance ofthe solubilized formazan was measured at 595 nm (OD₅₉₅peptide). For eachconcentration averages were calculated from triplicates. The percentageof growth was calculated as follows:(OD₅₉₅peptide-OD₅₉₅Tz)/(OD₅₉₅blank-OD₅₉₅Tz)×100%. The GI₅₀ (GrowthInhibition) concentrations were calculated for each peptide by using atrend line function for the concentrations (50, 25, 12.5 and 0 μM), thecorresponding percentages and the value 50, (=TREND (C₅₀:C₀,%₅₀:%₀,50).

2.5. Hemolysis

The peptides were tested for their hemolytic activity against human redblood cells (hRBC). Fresh hRBC were washed four times with phosphatebuffered saline (PBS) and centrifuged for 10 min at 3000×g. Compounds(100 μM) were incubated with 20% hRBC (v/v) for 1 h at 37° C. andshaking at 300 rpm. The final erythrocyte concentration wasapproximately 0.9×10⁹ cells/mL. A value of 0% and 100% cell lysis,respectively, was determined by incubation of hRBC in the presence ofPBS containing 0.00% acetic acid and 2.5% Triton X-100 in H₂O,respectively. The samples were centrifuged, the supernatants were 8-folddiluted in PBS buffer and the optical densities (OD) were measured at540 nm. The 100% lyses value (OD₅₄₀H₂O) gave an OD₅₄₀ of approximately0.5-1.0.

Percent hemolysis was calculated as follows:(OD₅₄₀peptide/OD₅₄₀H₂O)×100%.

2.6. Plasma Stability

The stability of the peptides in human and mouse plasma was determinedby applying the following method: 346.5 μL/deep well of freshly thawedhuman plasma (Basler Blutspende-dienst) and mouse plasma (HarlanSera-Lab, UK), respectively, were spiked with 3.5 μL/well of compounddissolved in DMSO/H₂O (90/10 [v/v], 1 mM, triplicate) and incubated at37° C. At t=0, 15, 30, 60, 120, 240 and 1440 min aliquots of 50 μL weretransferred to filtration plate wells containing 150 μL/well of 2%formic acid in acetonitrile. Following shaking for 2 min the occurredsuspensions were filtrated by vacuum. 100 μL of each filtrate weretransferred to a propylene microtiter plate and dried under N₂. Theresidual solids were reconstituted by adding 100 μL/well ofwater/acetonitrile, 95/5 (v/v)+0.2% formic acid and analyzed by LC/MS asfollows: Column: Waters, XBridge C18, mobile phases: (A) water+0.1%formic acid and (B) acetonitrile/water, 95/5 (v/v)+0.1% formic acid,gradient: 5%-100% (B) in 1.8 minutes, electrospray ionization, MRMdetection (triple quadrupole). The peak areas were determined andtriplicate values are averaged. The stability is expressed in percent ofthe initial value at t=0. (tx/t0×100). By using the TREND function ofEXCEL (Microsoft Office 2003) T1/2 were determined.

2.7. Plasma Protein Binding

495 μL aliquots of human plasma (Basler Blutspendedienst) as well as 495μL aliquots of PBS were placed in individual deepwells of apolypropylene plate (Greiner) and spiked each with 5 μL of 1 mMsolutions of peptides in 90% DMSO. After shaking the plate for 2 min at600 rpm 150 μL aliquots of the plasma/peptide mixtures were transferredin triplicates to the polypropylene filter plate (10 kDa, Millipore)whereas 150 μL aliquots of the PBS/peptide mixtures were transferredeither to the individual wells of the filter plate (filtered controls)or directly into the individual wells of the receiving plate (Greiner)(non-filtered controls). The plate sandwich consisting of filter andreceiving plate was incubated for 1 h at 37° C. and subsequentlycentrifuged at 3220 g for 2 h at 15° C. The filtrates in the receivingplate were analysed by LC/MS as follows: Column: Waters, XBridge C18,mobile phases: (A) water+0.1% formic acid and (B) acetonitrile/water,95/5 (v/v)+0.1% formic acid, gradient: 5%-100% (B) in 1.8 minutes,electrospray ionization, MRM detection (triple quadrupole). The peakareas were determined and triplicate values are averaged. The binding isexpressed in percent of the filtered and non-filtered controls by100-(100×T_(1 h)/T_(ctr)). Finally the average of these values iscalculated.

The results of the experiments described under 2.3-2.7 are indicated inthe Tables 1, 2, 3 and 4 herein below.

2.8. Pharmacokinetic Study (PK)

For the compounds of Ex. 1 and Ex. 2 pharmacokinetic studies afterintravenous (i.v.) administration were performed.

30 grams (±20%) male CD-1 mice obtained from Charles River LaboratoriesDeutschland GmbH were used. The vehicle, phosphate buffered saline, wasadded to give a final concentration of 0.5 mg/mL of the compound. Thevolume was 2 mL/kg and the compound was injected to give a finalintravenous dose of 1 mg/kg. Approximately 300-400 μL of blood wasremoved under light isoflurane anesthesia by cardiac puncture atpredetermined time intervals (5, 15, 30 min and 1, 2, 3, 4, hours) andadded to heparinized tubes. Plasma was removed from pelleted cells uponcentrifugation and frozen at −80° C. prior to HPLC-MS analysis.

Preparation of Plasma Calibration- and Plasma Study-Samples

Aliquots of 50 μL each of mouse plasma of untreated aminals (“blank”mouse plasma) were spiked with known amounts of the compounds Ex. 1 andEx. 2 in order to obtain 10 plasma calibration samples for each compoundin the range 1-4000 ng/mL. Aliquots of 50 μL each of mouse plasma fromtreated animals were used as plasma study samples.

Extraction of Plasma Calibration- and Plasma Study-Samples

All plasma samples were spiked with an appropriate internal standard andextracted with acetonitrile containing 2% formic acid. Supernatants wereevaporated to dryness under nitrogen and the remaining solidsreconstituted in water+0.2% formic acid/acetonitrile 95/5 (v/v).

LC-MS/MS-Analysis

Extracts were then analyzed by reverse-phase chromatography (Acquity HSSC18 SB, 100×2.1 mm, 1.8 μm column, Waters), using the followingconditions: mobile phases: (A) water+0.1% formic acid/acetonitrile 95/5(v/v), (B) aceto-nitrile/water+0.1% formic acid 95/5 (v/v), gradient: 1%(B) 0-0.1 min, 40% (B) 0.1-2.5. The detection and quantification wasperformed by mass spectrometry, with electrospray interface in positivemode and selective fragmentation of analytes (4000 Q Trap massspectrometer, AB Sciex).

Pharmacokinetic Evaluation

PK parameters were calculated by WinNonLin™ software version 5.3(Pharsight-A Certara™ Company, Moutain View, Calif. 94041 USA) using aone-compartmental model analysis. PK parameters were determined byleast-square fitting of the model to the experimental data.

The results of the experiments described in 2.8 are indicated in Tables5a and 5b herein below.

2.9. Drug Loading Calculations Via Maintainance Dose Rate (Rate ofInfusion)

The drug load for an implant comprising a peptide of the invention wascalculated following the basic principles in pharmacokinetics (see alsoJ. Gabrielsson, D. Weiner, “Pharmacokinetics and Pharmaco-dynamics DataAnalysis: Concepts and Applications”, 4^(th) edition, SwedishPharmaceutical Press, Stockholm, Sweden, 2006) whereby the maintainancedose rate (rate of infusion, R_(in)) can be defined as the rate at whicha drug is to be administered to reach a steady state of a certain dosein the plasma. The maintainance dose rate can be expressed using thecorrelation R_(in) [g/(h*kg)]=CL_(iv) [L/(h*kg)]×C_(ss,eff) [g/L],wherein CL_(iv) is the clearance (i.v.-admin.) and C_(ss,eff) theeffective concentration of the drug in the plasma at steady stateconsidering an efficacy margin A: C_(ss,eff) [g/L]=A×(IC₅₀/f_(u))×MW[(Mol/L)*(g/Mol)]. Therefore, the total amount of a drug loaded into animplant providing for a constant effective concentration of that drug inthe plasma for a certain period of time in a subject of a certain bodyweight can be calculated by applying the following correlation:

Drug_(load) [g/subject]=R _(in) [g/(h*kg)]×duration [h]×BW [kg/subject].

The results of the calculations described in 2.9 are indicated in Table6 herein below and based on the data given in Tables 1, 4 and 5b.Further pre-conditions are an efficacy margin of A=3, a study durationof 672 h (28 days) and a body weight of a human suject of 70 kg. Theglomerular filtration rate (GFR) which mainly influences the clearanceof the peptide is highly dependent on the species. In general, the GFRof humans is averaged to be 107 mL/(h*kg) compared to the GFR of mousebeing 840 mL/(h*kg). Therefore, the CL_(iv)-mouse values indicated inTable 5b were allometrically scaled by 107 mL/(h*kg)/840 mL/(h*kg)=0.127before employed in the above described correlations.

TABLE 1 Ex. IC₅₀ [nM] ± SD, CXCR4 receptor 1 0.17 ± 0.1 2 0.32 ± 0.1

TABLE 2 Hemolysis Cytotoxicity at Hela Cells Cos-7 Cells 100 μM Ex. GI₅₀[μM] GI₅₀ [μM] [%] 1 >42 >50 1.0 2 >34 >50 1.0

TABLE 3 Plasma stability human pl. mouse pl. cpd left at cpd left athuman pl. 1440 min mouse pl. 1440 min Ex. T_(1/2) [min] [%] T_(1/2)[min] [%] 1 1440 100 1440 100 2 1440 87 1440 100

TABLE 4 Ex. Plasma protein binding [%] Fraction unbound, f_(u) 1 55 0.452 43 0.57

TABLE 5a Ex. 1 Ex. 2 i.v. route i.v. route Dose: 1 mg/kg Dose: 1 mg/kgNum. Num. Calc. of Calc. of Time Conc. anim. Conc. anim. [h] [ng/mL]pool. [ng/mL] pool. 0.083 1693 3 1250 3 0.25 1050 1 1207 3 0.5 749 3 9963 1 385 3 516 3 2 61 3 117 3 3 <LoQ 3 24 3 4 <LoQ 3 14 3 <LoQ belowLimit of Quantification

TABLE 5b i.v. route Ex. 1 Ex. 2 Dose [mg/kg] 1 1 V_(dis) [mL/kg] 532 681CL_(iv) [mL/h/kg] 984 684 AUC_(0-∞) [ng * h/mL] 1016 1461 C_(max)[ng/mL] 1881 1469 Half-life [h] 0.4 0.7

TABLE 6 CL_(iv), human Molecular Weight (allometric (salt free), scaled)Drug_(load) Ex. MW [g/Mol] [mL/h/kg] [mg] 1 2127.53 125 14.2 ± 8.4 22066.40 87 14.2 ± 4.8

1. A compound of the general formula cyclo(-Tyr¹-His²-Xaa³-Cys⁴-Ser⁵-Xaa⁶-^(D)Pro⁷-Xaa⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Pro¹⁶-), disulfide bond between Cys⁴ and Cys¹¹, and pharmaceutically acceptable salts thereof, wherein Xaa³ is Tyr; Tyr(Me); or Tyr(CF₃), Tyr(Me) is (2S)-2-amino-(4-methoxyphenyl)-3-propionic acid, Tyr(CF₃) is (2S)-2-amino-(4-trifluoromethoxyphenyI)-3-propionic acid, Xaa⁶ is Ala; or Acc Acc is 1-aminocyclopropane-carboxylic acid, Xaa⁸ is Orn(iPr), Orn(iPr) is (2S)-N^(ω)-isopropyl-2,5-diaminopentanoic acid, Xaa¹³ is Gln; or Glu, Xaa¹⁴ is Lys(iPr), Lys(iPr) is (2S)-N^(ω)-isopropyl-2,6-diaminohexanoic acid, Xaa¹⁵ is ^(D)Pro; or ^(D)Lys(iPr), ^(D)Lys(iPr) is (2R)-N^(ω)-isopropyl-2,6-diaminohexanoic acid, with the proviso that if Xaa⁶ is Ala, then Xaa¹⁵ is ^(D)Lys(iPr).
 2. cyclo(-Tyr¹-His²-Tyr³-Cys⁴-Ser⁵-Ala⁶-^(D)Pro⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^(D)Lys(iPr)¹⁵-pro¹⁶-), disulfide bond between Cys⁴ and Cys¹¹, and pharmaceutically acceptable salts thereof.
 3. cyclo(-Tyr¹-His²-Tyr³-Cys⁴-Ser⁵-Acc⁶-^(D)Pro⁷-Orn(iPr)⁸-Arg⁹-Tyr¹⁰-Cys¹¹-Tyr¹²-Gln¹³-Lys(iPr)¹⁴-^(D)Pro¹⁵-Pro¹⁶-), disulfide bond between Cys⁴ and Cys¹¹, and pharmaceutically acceptable salts thereof.
 4. A compound according to claim 1 for use as therapeutically active substances, particularly as substances having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity.
 5. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically inert carrier, particularly in a form suitable for oral, topical, transdermal, injection, buccal or transmucosal administration such as a tablet, dragee, capsule, solution, liquid, gel, plaster, cream, ointment, syrup, slurry, suspension, powder or suppository.
 6. The use of a compound according to claim 1 as a medicament having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity, particularly for preventing HIV infections in healthy individuals; for slowing, or halting, the viral progression in an HIV infected patient; for treating or preventing, a cancer, or an immunological disease, that is mediated by, or results from, CXCR4 receptor activity; for treating immunosuppression; for accompanying the apheresis collection of peripheral blood stem cells; or for inducing the mobilization of stem cells to regulate tissue repair.
 7. The use of a compound according to claim 1 for the manufacture of a medicament having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity, particularly for preventing HIV infections in healthy individuals; for slowing, or halting, the viral progression in an HIV infected patient; for treating or preventing, a cancer, or an immunological disease, that is mediated by, or results from, CXCR4 receptor activity; for treating immunosuppression; for accompanying the apheresis collection of peripheral blood stem cells; or for inducing the mobilization of stem cells to regulate tissue repair.
 8. A process for the manufacture of compounds according to claim 1 which process comprises (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of Pro which in the desired end-product is in position 16; (b) removing the N-protecting group from the product thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position 15, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d) removing the N-protecting group from the product obtained in step (c); (e) effecting steps substantially corresponding to steps (c) and (d) using appropriately N-protected derivatives of amino acids which in the desired end-product are in positions n-2 to 1, any functional group(s) which may be present in said N-protected amino acid derivatives being likewise appropriately protected; (f) if desired, forming a disulfide bridge between the side-chains of the Cys residues at P4 and P11; or alternatively, forming the aforesaid linkage subsequent to step (i), as described herein below; (g) detaching the product thus obtained from the solid support; (h) cyclizing the product cleaved from the solid support; (i) removing any protecting groups present on functional groups of any members of the chain of amino acid residue; and (j) if desired, attaching one or several isopropyl groups (k) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound or into a different, pharmaceutically acceptable, salt.
 9. A compound according to claim 2 for use as therapeutically active substances, particularly as substances having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity.
 10. A compound according to claim 3 for use as therapeutically active substances, particularly as substances having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity.
 11. A pharmaceutical composition comprising a compound according to claim 2 and a pharmaceutically inert carrier, particularly in a form suitable for oral, topical, transdermal, injection, buccal or transmucosal administration such as a tablet, dragee, capsule, solution, liquid, gel, plaster, cream, ointment, syrup, slurry, suspension, powder or suppository.
 12. A pharmaceutical composition comprising a compound according to claim 3 and a pharmaceutically inert carrier, particularly in a form suitable for oral, topical, transdermal, injection, buccal or transmucosal administration such as a tablet, dragee, capsule, solution, liquid, gel, plaster, cream, ointment, syrup, slurry, suspension, powder or suppository.
 13. A pharmaceutical composition comprising a compound according to claim 4 and a pharnaceutically inert carrier, particularly in a form suitable for oral, topical, transdermal, injection, buccal or transmucosal administration such as a tablet, dragee, capsule, solution, liquid, gel, plaster, cream, ointment, syrup, slurry, suspension, powder or suppository.
 14. The use of a compound according to claim 2 as a medicament having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity, particularly for preventing HIV infections in healthy individuals; for slowing, or halting, the viral progression in an HIV infected patient; for treating or preventing, a cancer, or an immunological disease, that is mediated by, or results from, CXCR4 receptor activity; for treating immunosuppression; for accompanying the apheresis collection of peripheral blood stem cells; or for inducing the mobilization of stem cells to regulate tissue repair.
 15. The use of a compound according to claim 3 as a medicament having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity, particularly for preventing HIV infections in healthy individuals; for slowing, or halting, the viral progression in an HIV infected patient; for treating or preventing, a cancer, or an immunological disease, that is mediated by, or results from, CXCR4 receptor activity; for treating immunosuppression; for accompanying the apheresis collection of peripheral blood stem cells; or for inducing the mobilization of stem cells to regulate tissue repair.
 16. The use of a compound according to claim 4 as a medicament having CXCR4 antagonizing, anti-cancer activity and/or anti-inflammatory activity and/or stem cell mobilizing activity, particularly for preventing HIV infections in healthy individuals; for slowing, or halting, the viral progression in an HIV infected patient; for treating or preventing, a cancer, or an immunological disease, that is mediated by, or results from, CXCR4 receptor activity; for treating immunosuppression; for accompanying the apheresis collection of peripheral blood stem cells; or for inducing the mobilization of stem cells to regulate tissue repair.
 17. A process for the manufacture of compounds according to claim 2 which process comprises (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of Pro which in the desired end-product is in position 16; (b) removing the N-protecting group from the product thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position 15, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d) removing the N-protecting group from the product obtained in step (c); (e) effecting steps substantially corresponding to steps (c) and (d) using appropriately N-protected derivatives of amino acids which in the desired end-product are in positions n-2 to 1, any functional group(s) which may be present in said N-protected amino acid derivatives being likewise appropriately protected; (f) if desired, forming a disulfide bridge between the side-chains of the Cys residues at P4 and P11; or alternatively, forming the aforesaid linkage subsequent to step (i), as described herein below; (g) detaching the product thus obtained from the solid support; (h) cyclizing the product cleaved from the solid support; (i) removing any protecting groups present on functional groups of any members of the chain of amino acid residue; and (j) if desired, attaching one or several isopropyl groups (k) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound or into a different, pharmaceutically acceptable, salt.
 18. A process for the manufacture of compounds according to claim 3 which process comprises (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of Pro which in the desired end-product is in position 16; (b) removing the N-protecting group from the product thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position 15, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d) removing the N-protecting group from the product obtained in step (c); (e) effecting steps substantially corresponding to steps (c) and (d) using appropriately N-protected derivatives of amino acids which in the desired end-product are in positions n-2 to 1, any functional group(s) which may be present in said N-protected amino acid derivatives being likewise appropriately protected; (f) if desired, forming a disulfide bridge between the side-chains of the Cys residues at P4 and P 11; or alternatively, forming the aforesaid linkage subsequent to step (i), as described herein below; (g) detaching the product thus obtained from the solid support; (h) cyclizing the product cleaved from the solid support; (i) removing any protecting groups present on functional groups of any members of the chain of amino acid residue; and (j) if desired, attaching one or several isopropyl groups (k) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound or into a different, pharmaceutically acceptable, salt.
 19. A process for the manufacture of compounds according to claim 4 which process comprises (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of Pro which in the desired end-product is in position 16; (b) removing the N-protecting group from the product thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position 15, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d) removing the N-protecting group from the product obtained in step (c); (e) effecting steps substantially corresponding to steps (c) and (d) using appropriately N-protected derivatives of amino acids which in the desired end-product are in positions n-2 to 1, any functional group(s) which may be present in said N-protected amino acid derivatives being likewise appropriately protected; (f) if desired, forming a disulfide bridge between the side-chains of the Cys residues at P4 and P11; or alternatively, forming the aforesaid linkage subsequent to step (i), as described herein below; (g) detaching the product thus obtained from the solid support; (h) cyclizing the product cleaved from the solid support; (i) removing any protecting groups present on functional groups of any members of the chain of amino acid residue; and (j) if desired, attaching one or several isopropyl groups (k) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound or into a different, pharmaceutically acceptable, salt. 